Staphylococcus aureus has historically been a leading cause of nosocomial infections in humans worldwide. In the past several decades the emergence of methicillin-resistant S. aureus (MRSA) harboring multiple antibiotic resistance determinants has left relatively few therapeutic options available. Today, these strains have made their way into the community and now pose a very serious public health threat, causing more deaths per year than HIV-AIDS. The University of Nebraska Medical Center has committed to advancing research into the staphylococci with the hiring of several new researchers, and the concerted collaboration with regional investigators, who are all focused on this important pathogen. These researchers have expertise in varied aspects of the staphylococci including: biofilm development, gene regulation, physiology, and the immunology of staphylococcal infections. The hypothesis to be tested by this team in this application is that S. aureus biofilm formation involves complex developmental processes that affect the host immune response. The application includes four projects centered around staphylococcal biofilm and disease including: regulated cell death during biofilm development (Project 1, K. Bayles);effect of arginine metabolism on biofilm formation in the staphylococci (Project 2, P. Fey);the role of nuclease in biofilm development and disease (Project 3, A. Horswill);and innate immunity to S. aureus biofilm (Project 4, T. Kielian). The cores include: Biofilm Growth and Analysis Core (Core A, J. Bose);Bioimaging Core (Core B, T. Fritz);and Administrative Core (Core C, K. Bayles). Each project involves highly collaborative and synergistic research endeavors and relies heavily on the cores. These efforts will have a dramatic impact on our understanding of biofilm formation in the staphylococci and the effect it has on the host response. Ultimately, these studies will pave the way for novel therapeutic approaches for the treatment of staphylococcal infections. PROJECT 1: [Regulated cell death during biofilm development (Bayles, K)] PROJECT 1 DESCRIPTION (provided by applicant): Bacterial physiology has been extensively studied in the context of cell growth, but the molecular details by which bacteria undergo cell death and lysis have remained a near complete mystery. A growing body of recent evidence suggests that bacterial cell death and lysis involves active, genetically-encoded mechanisms that are critical to complex developmental processes such as sporulation and biofilm formation. The Staphylococcus aureus cid and Irg operons encode novel proteins that regulate bacterial death and lysis. CidA and LrgA proteins are proposed to be structurally and functionally similar to bacteriophageencoded holins and antihollns, and the ubiquitous distribution of these genes among bacteria suggests that they play a conserved physiological role. Recent studies have demonstrated an important biological role for CidA-mediated cell lysis during biofilm development, but the specific metabolic and environmental cues that regulate cid and /rg-mediated cell death and lysis within the context of biofilm growth remain ill-defined. Low oxygen growth and endogenous nitric oxide (NO) production have both been implicated as regulators of cell death and dispersal in biofilm of other bacteria, but the molecular mechanisms involved in these processes are not well understood. Preliminary data have suggested that growth under low oxygen conditions and NO are both potent signals that regulate cid and Irg expression. The scdA and NO-reductase {nor) genes, involved in the nitrosative stress response and NO metabolism, respectively, are also located in close proximity to lytSR-lrgAB in the clinical isolate UAMS-1. Thus, the central hypothesis of this application is that the transition into anaerobic metabolism during S. aureus biofilm growth is an important developmental signal in the control of Cid-/Lrg-mediated cell death and lysis. The specific aims of this project are 1) to study the transition to anaerobic metabolism during biofilm development and its effect on cid and Irg expression, 2) to examine the role of NO during biofilm development, and 3) to determine the molecular mechanism and role of LytSR-mediated regulation of cid and Irg expression during biofilm growth. Temporal and spatial patterns of aerobic and anaerobic metabolism within the biofilm will be determined using fluorescent reporter genes fused to aerobic and anaerobic promoters, and cid and Irg expression within these defined regions will be measured by laser capture microdissection microscopy (LCM) and real-time RT-PCR. The effect of NO donors and scavengers on cell death during biofilm development will be monitored using fluorescent dyes, and the effect of these compounds on cid and Irg expression will also be assessed. A detailed molecular characterization of the LytSR signal transduction cascade will also be performed to elucidate the role of this regulatory system during I0W-O2 growth and biofilm development. Collectively, these studies will reveal new insights into the molecular control of cell death and lysis during biofilm development.